Hamza et al. Bull. Iraq nat. Hist. Mus. (2020) 16 (1): 63- 81. https://doi.org/10.26842/binhm.7.2020.16.1.0063 A STUDY OF PHYSICAL AND ANATOMICAL CHARACTERISTICS OF THE HEAVY METAL ACCUMULATION OF JUNCUS RIGIDUS DESFONTAINES, 1798 (FAMILY, JUNCACEAE) IN BASRAH PROVINCE, SOUTHEREN OF IRAQ Shatha Mohammed Hamza* Sahar A. A. Malik Al-Saadi* and Dunya A. Hussain Al-Abbawy** * College of Science, Department of Biology, Basrah University, Basrah, Iraq ** College of Science, Department of Ecology, Basrah University, Basrah, Iraq *Corresponding author: [email protected] Received Date: 07 January 2020, Accepted Date: 11 April 2020, Published Date: 24 June 2020 ABSTRACT This study was carried out to determine the heavy metal accumulation of Juncus rigidus Desfontaines, 1798 from three different regions of the Basrah Province in Southern of Iraq. Specifically, the concentrations of lead, nickel, and cadmium were determined in the roots, culms and leaves of the plant. The results indicated that the highest accumulation of the heavy metal was recorded in lead (Pb) 12.50± 3.58 mg kg-1and then in nickel (< 0.30). The lowest value was recorded for cadmium (< 0.05). As well, lead concentrations in J. rigidus varied in different locations and parts of the plant from undetectable in control to 12.66, 19.33, and 9.80 mg kg-1 in leaves, culm, and roots respectively from Station 2, and 10.76, 12.66, and 9.50 mg kg-1 in Station 3. The values of translocation factor (TF), bioconcentration factor (BCF), and Biological Accumulation Coefficient (BAC) were greater than>1 used to the ability of J. rigidus for both phytoextraction and phytostabilization. The anatomical analysis showed that heavy metal accumulation in plant tissues led to a reduction in root and culm thickness; in polluted area it has been found that cortex and intercellular spaces in aerenchyma layers were deceased in size, whereas high pollution levels were observed in vascular bundles, which were smaller, and had increased sclerenchyma, as well as appeared more black or dark color compared to the specimens grown in the control area. Keyword: Accumulation, Anatomy, Culm, Heavy metals, Juncus rigidus, Root. 63 A study of physical and anatomical characteristics INTRODUCTION The genus of Juncus Linnaeus, 1753 belongs to the family of Juncaceae, which has approximately 250 - 300 species worldwide. Among the flora of Iraq, this genus has six subgenera (Juncus, Genuini, Subulati, Pseudotenageia, Poiophylli and Septati), is widely distributed, and has 16 species. Juncus, commonly called rushes or samar, is a perennial plant; all of its leaves are basal; terete, pungent, auricles are absent; flowers are in stalked panicles, and seeds contain appendages (Townsend and Guest, 1985). J. rigidus Desfontaines, 1798 grows in marshes, shallow brackish water, and semi-saline soil and can be found in a variety of moist, wet and temperate climates (Snogerup, 1978; Townsend and Guest, 1985). J. rigidus species are used in traditional medicine for their antioxidant, antimicrobial, antitumor, cytotoxic, antiviral, anti-algal, and anti-inflammatory properties (El-Shamy et al., 2015). This genus contains several medically relevant compounds including terpenes, flavonoids, phenolic acids, coumarins, sterols, carotenoids, stilbenes and phenanthrenes. Seeds of this plant are rich in fatty acids and amino acids (Osman et al., 1975; Zahran and El- Habib, 1979). Heavy metals are some of the most critically relevant environmental pollutants (Tangahu et al., 2011); the metals sources include natural rock erosion and human activities, for example, industrial processes go into unpolluted areas where they accumulate in the water, soil, deep sediment, and living organisms (Miretzky et al., 2004). Utilizing plants to remove this form of pollutants have been investigated since the early 1970s (Susarla et al., 2002; Bouldin et al., 2006). Many plant species are well suited for phytoremediation due to their ability to absorb heavy metals such as Pb, Cd, Cr, Ag, and various radionuclides from soil (Lasat, 2000). Phytoremediation can remove heavy metals many (e.g., Fe, Mn, Zn, Cd, Cr, Pb, Co, Ag, Se, Hg, Cu, Mg, Mo, and Ni) (Cho-Ruk et al., 2006); this technique uses plants to accumulate pollution, is affordable, and is environmentally friendly (Najeeb et al., 2017). Collection of plant species for phytoremediation purposes is related to, or depends on plants being in open biological systems with the latent to accumulate more heavy metal of dry biomass and growth rates, (Susarla et al., 2002; McGrath and Zhao, 2003). Plant uptakes of pollution from soil particles or soil liquid via root systems, and from cracks down of polluted sites from soils, sediments and water, and then they go through translocation and bioaccumulation to the internal plant structure (Cho-Ruk et al., 2006; Paz-Alberto and Sigua, 2013). Many species of Juncus are used as accumulators; Juncus effuses Linnaeus, 1753, is one of the 17 terrestrial species that has ability to accumulate high concentrations of heavy metals like Pb, Cd, Cu, and Zn. In addition, to removes phyto-stabilization in wetlands through genetic manipulation (Yanqun et al., 2004; Grube et al., 2008; Najeeb et al., 2011; Najeeb et al., 2017). This species was tolerant to stress from heavy metals such as zinc and chromium (Dimitroula et al., 2014; Mateos-Naranjo et al., 2014). 64 Hamza et al. The study area (Basrah City) contains developed industrial or urban regions which led to many environmental problems, and caused an increase in pollution, including heavy metals (Al-Obaidy et al., 2016). Many studies have evaluated and identified the sediment and water of the most pollution sites (Akesh, 2017). The influential heavy elements (Pb, Cd, Cr and Ni) are increasing in Basrah city because of its closeness to oil companies or industrial waste regions which contain high levels of metals such as oil refinery of Al-Sha’eiba (Khwedim et al., 2009). The purpose of the present study was to determine the bioaccumulation of three heavy metals Pb, Cd, and Ni in the roots, culm, and leaves of J. rigidus, which have grown in different contaminated sites in Basrah in order to determine the applicability of J. rigidus for phytoremediation, and to observe the anatomical changes of roots and culm structure. MATERIALS AND METHODS Study area and sampling Experiments were conducted at the College of Science, Department of Biology and Ecology, Basrah University. Three stations were selected: Station 1 included a control of uncontaminated area (Garmat Ali) Global Positioning System (GPS) 47◦ 45´ 46´´ E 30◦ 34´ 46´´N; Station 2 near oil contamination (Al-Sha’eiba) 47◦ 41´ 59´´ E 30◦ 22´ 59´´N; and Station 3 near energy engine contamination (Taga place) 47◦ 45´ 58´´ E 30◦ 39´ 44´´N. J. rigidus specimens were collected in the summer of 2017 from the three stations and brought into the laboratory on the same day. Then, to remove the remaining soil from the plant materials, the specimens were washed carefully three times with distilled water to remove adhering particles/ remaining soil and then were dried. Chemical analysis of the heavy metals (Cd, Ni, and Pb) in the roots, culm, and leaves of J. rigidus plant was achieved through HNO3 digestion and the final filtrated mixture was subjected to an atomic spectrophotometer (Phoenix-986, CITY, England). The concentration of heavy metals detected in the plant was determined through comparison to a standard curve of concentrations (Kabata-Pendias and Pendias, 1992); the soil samples were collected at 0 –15 cm depths, then the specimens dried in oven at 150 °C for 12 hours were digested in acid-cleaned Teflon microwave vessels hydrofluoric acid2ml and 5ml of nitric acid and they were digested at 200◦C for 30 min (Binning and Baird, 2001). Heavy metals content concentrations (Pb, Ni and Cd) were determined by using atomic spectrophotometer (Phoenix-986, CITY, England). The working wave lengths were as follows: Pb 217 nm; Cd -228.8 nm and Ni 232 nm and limited of detection for each element: Pb, Ni and Cd were 0.01 ppm. Phytoextraction efficiency Three parameters were calculated to compare the accumulation and translocation of heavy metals from the roots to the culms including: the bioconcentration factor (BCF), the translocation factor (TF), and the Biological Accumulation Coefficient (BAC) (Yoon et al., 2006). BCF is considered as the percentage of mineral concentration in roots to soil (Yoon et 65 A study of physical and anatomical characteristics al., 2006). TF reflects the proportion of the heavy metals in the shoot to its roots whereas the BAC explains ratio of the heavy metals in the shoots to the soil (Cui et al., 2007; Li et al., 2007) as the following: BCF = (Concentration Metals) root / (Concentration Metals) soil TF = (Concentration Metals) shoot / (Concentration Metals) root BAC = (Concentration Metals) shoot / (Concentration Metals) soil Anatomical study For the anatomical studies, ten specimens of J. rigidus plants were collected from each station; the permanent sections of roots and culms were ready, the plant parts were cut into 10-15 cm pieces and fixed for 24 hours in formalin-acetic acid and alcohol (FAA) and were preserved in 70% ethyl alcohol, then dehydrated in an ethyl alcohol series. Then, specimens were sectioned on a rotary microtome, and stained in safranin and fast green before being mounted in Canada balsam on glass slides (Johansan, 1968). 100 slides were prepared from J. rigidus specimens in each station. In this study, unpolluted and polluted plant parts from stations were analyzed; the five best transverse sections were selected to study anatomical features. Finally, the specimens were examined with an Olympus light microscope and photographed with DCE-2 digital camera (Metcalfe and Chalk, 1950; Esau, 1977). Statistical analysis The data of the study were analyzed by one-way analysis of variance (ANOVA).